Keratinocyte stem cells have an unquestioned role in maintaining the normal structure and function of the epidermis and hair follicles and are thought to be important players in inherited and acquired skin disease. Hence, identification of genes regulating their number and proliferative potential is a critical problem in cutaneous biology. We propose here a novel strategy for identifying genes involved in keratinocyte stem cell regulation. This strategy takes advantage of several recent important advances made in our laboratory: 1) identification of a selectable marker on hair follicle stem cells, 2) development of a sensitive and quantitative in vitro assay for clonogenic keratinocyte stem cells, 3) genetic mapping of several loci with linkage to stem cell number analysis of stem cell gene expression, and 4) analysis of keratinocyte stem cell gene expression. The objective of this proposed research is to identify major genes regulating the number of keratinocyte stem cells. Our hypothesis is that there are specific genes and pathways that regulate the number of keratinocyte stem cells that may be different from those regulating transit-amplifying cells The focus of Specific Aim 1 is to use genetic tools to refine the linkage intervals we have already identified. In Specific Aim 2 we will use a candidate gene approach for stem cell gene identification;including the functional significance of the sequence variant in the Bmps gene already identified. In Aim 3, we will use a complementary global genomic approach to identify associated molecular pathways, and to assess regulatory polymorphisms causing differences in gene expression in the absence of coding sequence differences within the quantitative trait locus. The investigation we detail in this proposal will enable the identification of major genes and regulatory pathways in keratinocyte stem cells. This research will impact the fields of cutaneous biology and stem cell research and should provide new insight into the mechanism of skin carcinogenesis. Identification of stem cell regulatory genes is important for gene therapy as well as for the design of new therapeutic modalities for chronic hyperproliferative skin disease, for wound that do not heal, and for skin cancer.